The present invention relates generally to compositions and methods for hybridization of oligonucleotides, and more specifically to certain solutions and/or oligonucleotide analogues which may increase hybridization and priming specificity.
The detection of diseases is increasingly important in prevention and treatments. While multifactorial diseases are difficult to devise genetic tests for, more than 200 known human disorders are caused by a defect in a single gene, often a change of a single amino acid residue (Olsen, Biotechnology: An industry comes of age, National Academic Press, 1986). Many of these mutations result in an altered amino acid that causes a disease state.
Sensitive mutation detection techniques offer extraordinary possibilities for mutation screening. For example, analyses may be performed even before the implantation of a fertilized egg (Holding and Monk, Lancet 3:532, 1989). Increasingly efficient genetic tests may also enable screening for oncogenic mutations in cells exfoliated from the respiratory tract or the bladder in connection with health checkups (Sidransky et al., Science 252:706, 1991). Also, when an unknown gene causes a genetic disease, methods to monitor DNA sequence variants are useful to study the inheritance of disease through genetic linkage analysis. However, detecting and diagnosing mutations in individual genes poses technological and economic challenges. Several different approaches have been pursued, but none are both efficient and inexpensive enough for truly widescale application.
Mutations involving a single nucleotide can be identified in a sample by physical, chemical, or enzymatic means. Generally, methods for mutation detection may be divided into scanning techniques, which are suitable to identify previously unknown mutations, and techniques designed to detect, distinguish, or quantitate known sequence variants.
Several scanning techniques for detection of mutations have been developed on the observation that heteroduplexes of mismatched complementary DNA strands exhibit an abnormal behavior, especially when denatured. This phenomenon is exploited in denaturing and temperature gradient gel electrophoresis (DGGE and TGGE, respectively) methods. Duplexes mismatched in even a single nucleotide position can partially denature, resulting in retarded migration, when electrophoresed in an increasingly denaturing gradient gel (Myers et al., Nature 313:495, 1985; Abrams et al., Genomics 7:463, 1990; Henco et al., Nucl. Acids Res. 18:6733, 1990). Although mutations may be detected, no information is obtained regarding the precise location of a mutation. Mutant forms must be further isolated and subjected to DNA sequence analysis.
Alternatively, a heteroduplex of an RNA probe and a target strand may be cleaved by RNase A at a position where the two strands are not properly paired. The site of cleavage can then be determined by electrophoresis of the denatured probe. However, some mutations may escape detection because not all mismatches are efficiently cleaved by RNase A.
Mismatched bases in a duplex are also susceptible to chemical modification. Such modification can render the strands susceptible to cleavage at the site of the mismatch or cause a polymerase to stop in a subsequent extension reaction. The chemical cleavage technique allows identification of a mutation in target sequences of up to 2 kb and it provides information on the approximate location of mismatched nucleotide(s) (Cotton et al., PNAS USA 85:4397, 1988; Ganguly et al., Nucl. Acids Res. 18:3933, 1991). However, this technique is labor intensive and may not identify the precise location of the mutation.
An alternative strategy for detecting a mutation in a DNA strand is by substituting (during synthesis) one of the normal nucleotides with a modified nucleotide, thus altering the molecular weight or other physical parameter of the product. A strand with an increased or decreased number of this modified nucleotide relative to the wild-type sequence exhibits altered electrophoretic mobility (Naylor et al., Lancet 337:635, 1991). This technique detects the presence of a mutation, but does not provide the location.
Two other strategies visualize mutations in a DNA segment by altered gel migration. In the single-strand conformation polymorphism technique (SSCP), mutations cause denatured strands to adopt different secondary structures, thereby influencing mobility during native gel electrophoresis. Heteroduplex DNA molecules, containing internal mismatches, can also be separated from correctly matched molecules by electrophoresis (Orita, Genomics 5:874, 1989; Keen, Trends Genet. 7:5, 1991). As with the techniques discussed above, the presence of a mutation may be determined but not the location. As well, many of these techniques do not distinguish between a single and multiple mutations.
All of the above-mentioned techniques indicate the presence of a mutation in a limited segment of DNA and some of them allow approximate localization within the segment. However, sequence analysis is still required to unravel the effect of the mutation on the coding potential of the segment. Sequence analysis is a powerful tool, allowing, for example, screening for the same mutation in individuals of an affected family, monitoring disease progression in the case of malignant disease, or for detecting residual malignant cells in bone marrow before autologous transplantation. Despite these advantages, the procedure is unlikely to be adopted as a routine diagnostic method because of the high expense involved.
A large number of other techniques have been developed to analyze known sequence variants. Automation and economy are very important considerations for implementation of these types of analyses. In this regard, none of the alternative techniques discussed below combine economy and automation with the required specificity.
A number of strategies for nucleotide sequence distinction all depend on enzymes to identify sequence differences (Saiki, PNAS USA 86:6230, 1989; Zhang, Nucl. Acids Res. 19:3929, 1991).
For example, restriction enzymes recognize sequences of about 4-8 nucleotides. Based on an average G+C content, approximately half of the nucleotide positions in a DNA segment can be monitored with a panel of 100 restriction enzymes. As an alternative, artificial restriction enzyme recognition sequences may be created around a variable position by using partially mismatched PCR primers. With this technique, either the mutant or the wild-type sequence alone may be recognized and cleaved by a restriction enzyme after amplification (Chen et al., Anal. Biochem. 195:51, 1991; Levi et al., Cancer Res. 51:3497, 1991).
Another method exploits the property that an oligonucleotide primer that is mismatched to a target sequence at the 3xe2x80x2 penultimate position exhibits a reduced capacity to serve as a primer in PCR. However, some 3xe2x80x2 mismatches, notably G-T, are less inhibitory than others, thus limiting its usefulness. In attempts to improve this technique, additional mismatches are incorporated into the primer at the third position from the 3xe2x80x2 end. This results in two mismatched positions in the three 3xe2x80x2 nucleotides of the primer hybridizing with one allelic variant, and one mismatch in the third position in from the 3xe2x80x2 end when the primer hybridizes to the other allelic variant (Newton et al., Nucl. Acids Res. 17:2503, 1989). For this technique to be successful, it is necessary to define amplification conditions that significantly disfavor amplification in the presence of a 1 bp (basepair) mismatch. In fact, this technique is rarely successful (see, e.g., Sininsky, J. Nucl. Acids Res., 1990).
DNA polymerases have also been used to distinguish allelic sequence variants by determining which nucleotide is added to an oligonucleotide primer immediately upstream of a variable position in the target strand. Based on this approach, a ligation assay has been developed. In this method, two oligonucleotide probes hybridizing in immediate juxtaposition on a target strand are joined by a DNA ligase. Ligation is inhibited if there is a mismatch where the two oligonucleotide probes abut.
Mutations may be identified via their destabilizing effects on the hybridization of short oligonucleotide probes to a target sequence (see Wetmur, Crit. Rev. Biochem. Mol. Biol. 26:227, 1991). Generally, this technique, allele-specific oligonucleotide hybridization, involves amplification of target sequences and subsequent hybridization with short oligonucleotide probes. An amplified product can be scanned for many possible sequence variants by determining its hybridization pattern to an array of immobilized oligonucleotide probes. Many of these techniques, especially allele-specific oligonucleotide hybridization, require establishing conditions that favor the hybridization of an exact match over a mismatch. As is well known, such conditions are difficult to achieve. One approach to improving hybridization is the addition of a chaotrope.
Chaotropes decrease the melting temperature of an oligonucleotide duplex (see Van Ness and Chen, Nucleic Acids Research 19:5143, 1991). Oligonucleotide probes (12-50 mers) possess some functional properties that are not shared by long DNA probes. These parameters include different rates of duplex formation as a function of (a) the difference between the hybridization temperature and the Tm, (b) stringency requirements for maximal selectivity/specificity of hybridization, and (c) sequence-specific anomalous behavior.
Chaotropes are useful in DNA probe-based diagnostic assays, as they can simultaneously lyse the cells of organisms of interest, inhibit nucleases and proteases, and provide adequate hybridization stringency without chemically altering the target analyte. Chaotropic lysis and hybridization solutions eliminate the need to isolate nucleic acid prior to conducting the DNA probe assay, and facilitate the development of rapid and simple assay formats (see Van Ness and Chen, Nucleic Acids Research 19:5143, 1991, for review). However, the commonly used chaotropes do not substantially increase the differential hybridization of matched/mismatched sequences. Furthermore, they do not neutralize the dependence of Tm and Td on G+C content.
In addition, special problems arise when hybridization methods are employed that involve the use of mixed pools of oligonucleotide probes (12- to 50-mers) having differing base sequences and G+C content. Many applications utilize mixed pools of oligonucleotides and are frustrated by a host of problems. For example, many gene isolation strategies involve the reverse translation of a known polypeptide sequence into a set of all possible DNA sequences that can encode that protein (Jaye et al., Nucl. Acids Res. 11:2325-2335, 1983). A pool of oligonucleotide probes, homologous to the set of possible protein encoding DNA sequences, are then used to screen a genomic or cDNA library from the relevant organism or cell type in order to identify the desired gene sequence. While the length of all of the oligonucleotide probes is the same, the G+C content of each probe may vary significantly, making the selection of hybridization conditions that are suitable and specific for each oligonucleotide problematic. As a result, often many false positive clones will be selected when screening highly complex libraries for genes of low abundance.
This problem of simultaneously and accurately hybridizing many differing oligonucleotides of differing G+C content is even greater for sequence analysis of a specific region of DNA or identifying single base change mutations using large arrays of oligonucleotides (which may vary from 100% A+T to 100% G+C) bound to a fixed surface (Southern et al., Genomics 13:1008-1017, 1992; Maskos and Southern, Nucl. Acids Res. 20:1675-1678, 1992). These methods, while theoretically powerful, have been sorely limited by the inability to identify hybridization conditions that will facilitate accurate hybridization (i.e., no mismatch hybrid duplexes formed) and allow all possible perfect hybrids to be stably formed.
One attempted solution has been to use a class of salts composed of small alkylammonium ions (most commonly tetramethylammonium (TMA+) and tetraethylammonium (TEA+)), that can greatly decrease the effect of base composition on DNA melting (Marky et al., Biochemistry 20:1427-1431, 1981; De Murcia et al., Biophys. Chem. 8:377-383, 1978; Melchior and Von Hippel, Proc. Nat. Acad. Sci. USA 70:298-302, 1973). Of the tetraalkylammonium salts, only TMA+ and TEA+ are small enough to fit into the major groove of the B-form DNA double helix where they bind to the A+T base pairs of DNA (perhaps to the O-2 of thymine) (see De Murcia et al., Biophysical Chemistry 8:377 1978). The overall effect on stability is two-fold with the first being that the tetraalkylammonium salts increase the non-polar character of the solvent which destabilizes the base stacking interactions in native DNA (see Rees et al., Biochemistry 32:137, 1993). The second effect is that the A+T base pairs are stabilized. Specifically, TMACl prevents DNA premelting by decreasing the transient openings between the base pairs from occurring below the melting temperature (see De Murcia et al., Biophysical Chemistry 8:377 1978; Marky et al., Biochemistry 20:1427, 1981). The exact nature of TEACl stabilization is not known. Overall, the A+T pairing is stabilized resulting in a rise in the melting temperature for the A+T pairs (see Marky et al., Biochemistry 20:1427 1981; De Murcia et al., Biophysical Chemistry 8:377 1978). For 100% A+T oligonucleotide duplexes, the Tm in TMACl is actually 6xc2x0 C. higher than that found in a sodium solution (see Marky et al., Biochemistry 20:1427, 1981).
When genomic DNA is melted in TMACl or TEACl at the specific concentrations of 3 M and 2.4 M, respectively, identical melting temperatures are exhibited for A+T and G+C pairs (see Melchior et al., Proc. Natl. Acad. Sci. USA 70:298, 1973). Usually what is observed is that synthetic DNA duplex stability in concentrated TMACl and TEACl stability is somewhat diminished and has little base compositional dependence (see Wood et al., Proc. Natl. Acad. Sci. USA 82:1585 1985; Marky et al., Biochemistry 20:1427 1981; Jacobs et al., Nucleic Acids Res. 16:4637, 1988). For example, a series of 19-mers ranging from 26% G+C to 79% G+C content had melting temperatures over a range of 18xc2x0 C. in 2xc3x97SSC, while in 3 M TMACl the range narrowed to 5xc2x0 C. and in 2.4 M TEACl, the temperatures were virtually unchanged negating all influence from G+C content (see Jacobs et al., Nucleic Acids Res. 16:4637, 1988). TEACl had the added benefit of reducing the melting temperature approximately 22xc2x0 C. over TMACl and SSC (see Jacobs et al., Nucleic Acids Res. 16:4637, 1988). When various lengths of hybridization probes are measured and the corresponding melting temperatures plotted versus length, the plot is a smooth curve even though the G+C content varied from 31-66% (see Wood et al., Proc. Natl. Acad. Sci. USA 82:1585 1985).
In the context of gene isolation from complex libraries, the number of false positive clones isolated using a 17-mer mixed oligo pool (G+C range of 47% to 71%) was reduced 100-fold when performed in 3 M TMACl rather than using a NaCl hybridization solution (Wood et al., Proc. Nat. Acad. Sci. USA 82:1585-1588, 1985). However, even when using TMACl to eliminate the base composition effect on Tm, a significant number of false positive clones are still isolated due to formation of mismatched hybrids.
Using deoxyinosine at the third codon position (Honorxc3xa9 et al., J. Biochem. Biophys. Methods 27:39-48, 1993) of highly degenerate oligonucleotide pools from backtranslated protein sequences allows the oligonucleotide pool size to be significantly reduced. However, when screening a more complex genomic library for clones, the isolation of false positive clones may still be a significant problem (Jacobs et al., Nucl. Acids Res. 16:4637-4650, 1988). While the presence of tetramethylammonium and tetraethylammonium salts made oligonucleotide melting independent of base composition, there was no or little effect of mismatches on the thermal melting of oligonucleotides. That is, duplexes containing a mismatch had a similar Tm to duplexes which were perfectly base-paired.
Another method used to enhance specificity in hybridization reactions creates base mismatches using base analogs to replace any of the A, G, C, or T nucleotides. Research has shown that some primers containing a base pair mismatch have increased specificity when the mismatch is placed in precise locations (see Wenham et al., Clinical Chemistry 37:241, 1991; Newton et al., Nucleic Acids Research 17:2503, 1989; Ishikawa et al., Human Immunology 42:315, 1995). However, differences of as little as 0.5xc2x0 C. in the melting temperatures are equally common between perfectly matched hybrids and the same hybrid with a single base mismatch introduced (see Tibanyenda et al., European Journal of Biochemistry 139:19, 1984; Wemtges et al., Nucleic Acids Research 14:3773, 1986). Even better specificity has been noted between one and two base mismatched duplexes than has been observed between a perfectly matched duplex and the same duplex with a single mismatch (see Guo et al., Nature Biotechnology 15:331, 1997). Guo et al. found a (Tm of 4xc2x0 C. between zero and one mismatches and a xcex94Tm of 13xc2x0 C. between one and two adjacent mismatches for a 20-mer duplex. However, even with two mismatches, often there is still little destabilization of the duplex. This inability to consistently discriminate mismatches lends to the lack of specificity in PCR.
The use of more than one base pair mismatch per hybridization employing at least one nucleotide analog has been evaluated (see Guo et al., Nature Biotechnology 15:331, 1997). In this case, the analog compound consists of 3-nitropyrrole replacement of the purine or pyrimidine bases. 3-Nitropyrrole has the ability to minimally hydrogen bond with all four bases (see Nichols et al., Nature 369:492, 1994; Bergstrom et al., Journal of the American Chemical Society 117:1201, 1995). By introducing an artificial mismatch, large differences in the duplex melting temperatures occur ranging from approximately 5xc2x0 C. to 15xc2x0 C. with the largest difference occurring when the mismatch is located at the center of the 15-mer hybridizing oligo. Significant differences in xcex94Tm occur when an artificial nucleotide is introduced into a duplex that already contains a base mismatch creating a two-mismatch duplex. The degree of destabilization depends upon the type of base mismatch (e.g., G/T) and the separation between the two mismatches. In experimental examination, the base analog nucleotide ranged from 1 to 7 bases to the 3xe2x80x2 side of the base mismatch, which was held in the center of the 15-mer. Differences in xcex94Tm for the three different base mismatched 15-mers ranged from a 2xc2x0 C. stabilization (in the C/T mismatch case only and when the mismatches are adjacent) to a 7xc2x0 C. further destabilization with the maximum destabilization consistently occurring at a 3 or 4 base mismatch separation (see Guo et al., Nature Biotechnology 15:331, 1997).
When two artificial mismatches are introduced, the proximity of the artificial bases greatly influences the degree of destabilization. The two artificial mismatches were centered on the middle of a 21-mer duplex beginning with a separation of 6 bp. The destabilization, or xcex94Tm, is minimally 12xc2x0 C. when compared to the perfectly matched duplex. The greatest difference of over 20xc2x0 C. occurs when the two artificial mismatches are 10 base pairs apart. This difference corresponds to one helical turn and indicates that some kind of interaction occurs between the two artificial bases that decreases the stability of the duplex.
Experimentally, when the PCR primer utilized contained one or two artificial mismatches between the primer and the DNA sample, the PCR gave results as would be expected for a perfectly matched primer (see Guo et al., Nature Biotechnology 15:331, 1997). However, when the primer contained both a true and an artificial mismatch, the PCR failed to produce any measurable results; while PCR with perfectly matched and true mismatches all produced measurable amounts of PCR product. The same study found similar results when using hybridization probes: those with perfect matches, true mismatches and artificial mismatches annealed while the probes containing artificial and true mismatches did not. These studies indicate greater ispecificity is created when artificial base mismatches are incorporated in hybridization reactions such that when naturally occurring mismatches occur, they are thermodynamically less stable than a perfectly matched hybridization reaction and thus less likely to produce a false positive in an assay or PCR. Interestingly, however, the difference in thermodynamic stability noted above for duplexes containing only artificial mismatches is not manifested in the experimental situation.
A further means of effecting hybridization discrimination is through differences in the stability between hybridization duplexes that contain nicks and gaps. In these reactions, duplexes are formed from tandernly stacked short oligomers hybridized to a longer strand that either align contiguously or non-contiguously leaving a few base pair gap. Hybridizations that result in a nick are subject to xe2x80x9cstacking hybridizationxe2x80x9d where another DNA strand hybridizes across the nick site. Stacking hybridization does not occur where gaps are present in the non-contiguous oligomers. The stacking has the effect of increased discrimination as evidenced by decreased dissociation rates and greater thermodynamic stability than the non-contiguous counterparts (see Lane et al., Nucleic Acids Res. 25:611, 1997). Thermodynamic measurements show differences between the hybridization stacked duplexes standard free energy change (xcex94G) and the gapped duplexes is 1.4 to 2.4 kcal/mol. Therefore, discrimination in hybridization can be afforded through the use of multiple short probes.
Most of the base mimics in current use are the result of the pursuit for a universal base. Many utilize nitroazole base analogues and have demonstrated reduced discrimination in base pairing. A series of nitroazole nucleobase analogues have been studied in attempts to gain additional insight into the significance of electronic structure and heterocyclic size in base pairing for the development of more effective universal bases (see Bergstrom et al., Nucleic Acids Res. 25:1935, 1997). In this work, the thermodynamic properties of the deoxyribonucleosides of 3-nitropyrrole, 4-nitropyrazole, 4-nitroimidazole, and 5-nitroindole were measured. For comparison, thermodynamic measurements were also made on the deoxyribonucleosides of hypoxanthine and pyrazole as well an abasic spacer, 1,2-dideoxyribose. Four oligonucleotides were synthesized for each modified nucleoside in order to obtain duplexes in which each of the four natural bases was placed opposite the base mimic. All of the base mimics analyzed proved to be far less stable than the natural base pairings (A+T: Tm=65.7xc2x0 C., C+G: Tm=70.5xc2x0 C.) with the Tms ranging from 35-46xc2x0 C. for 5-nitroindole to 18-29xc2x0 C. for the other nitroazole bases analyzed. The only exception was 4-nitroimidazole paired with dGTP where the Tm was 40.9xc2x0 C. In analyzing the free energy for the duplex melting, the 3-nitropyrrole base mimic was found to have the least discrimination when pairing with any of the four naturally occurring bases with an overall xcex94G of 0.4 kcal/mol. The next least discriminating was 5-nitroindole with a xcex94G of 0.8 kcal/mol. Both of these values are less than the xcex94G of 1.1 kcal/mol found between the natural base pairings of A+T and G+C. 4-Nitropyrazole showed a slight preference for pairing with A with a xcex94G of 1 kcal/mol more stable than C, G, and T free energies. Finally, 4-nitroimidazole showed a high selectivity for pairing to G (as was evidenced by its high Tm value) due to the ability of the imidazole N3 to hydrogen bond with the deoxyguanosine N1. It should be noted, however, that the above values are dependent upon the nearest base neighbors to the mimic. Further studies altered the nearest neighbors and found that 3-nitropyrrole and 5-nitroindole are quite non-discriminating base pairing partners.
Of interest, the enthalpy and entropy changes were found to track one another (i.e., a large enthalpy change correlates to a large entropy change) regardless of the base mimic utilized implying that the correlation between AS and AH is independent of the mode of association of the bases. What was observed was that small enthalpy and entropy changes were found in the non-hydrogen bonding base mimics. The low values for entropy change reflect the greater degree of freedom of movement possible for bases that are not locked into the duplex by hydrogen bonding interactions. The small enthalpy changes reflect alterations in hydrogen bonding interactions as a result of the loss of hydrogen bonding interactions for the base opposite the base mimic. If a natural base remains stacked in the helix without an opposing hydrogen bonding partner then it has lost hydrogen bonding interactions with water without regaining a new donor/acceptor partner.
A similar study involved examining acyclic nucleoside analogues with carboxamido- or nitro-substituted heterocyclic bases (see Aerschot et al., Nucleic Acids Res. 23:4363, 1995). Utilization of acyclic nucleosides endows the constructs with enough flexibility to allow good base stacking as well as allow the base mimics to obtain an orientation to best base-pair with the corresponding base. The heterocyclic bases examined included: 4,5-imidazoledicarboxamide, 4-nitroimidazole, and 5-nitroindazole. These complexes were referenced against acyclic hypoxanthine, 1-(2(-deoxy-(-D-ribofuranosyl)-3-nitropyrrole, 5-nitroindole, and 2-deoxyinosine. All the new acyclic complexes had melting temperatures 7-20xc2x0 C. less than those observed for the natural bases. 5-Nitroindazole when paired against each of the four natural bases had the least spread in xcex94Tm of only 2.2xc2x0 C. while the 4-nitroimidazole had a spread of 8.0xc2x0 C. with dG being significantly out of line with the other three bases as had similarly been observed above. Of the reference compounds, deoxyinosine had a xcex94Tm of 5.6xc2x0 C., 5-nitroindole""s xcex94Tm was 1.0xc2x0 C., 1-(2(-deoxy-(-D-ribofuranosyl)-3-nitropyrrole had a xcex94Tm of 5.1xc2x0 C., and the xcex94Tm of acyclic hypoxanthine was 4.8xc2x0 C. However, all base mimics showed about the same destabilization (xcex94Tm of 4-5xc2x0 C.) when placed in an oligo consisting almost exclusively of adenosines with exception of 4-nitroimidazole and acyclic deoxyinosine that had xcex94Tms of 7.0xc2x0 C. and 8.9xc2x0 C., respectively.
Aerschot and co-workers also examined the effect of incorporation of multiple base mimics into an oligo (see Aerschot et al., Nucleic Acids Res. 23:4363, 1995). Overall, melting temperatures dropped but most markedly with the incorporation of three base mimics. The nitroindoles, however, showed the least amount of temperature differential.
Another base mimic, 1-(2(-deoxy-(-D-ribofuranosyl)imidazole-4-carboxamide (Nucleoside 1), mimics preferentially dA as well as dC nucleosides (see Johnson et al., Nucleic Acids Res. 25:559, 1997). The ability to substitute for both dA and dC results from rotation about the carboxamide/imidazole bond as well as the bond between the imidazole and furanose ring. When the imidazole is anti to the furanose and the carboxamide group is anti to the imidazole, the lone pair on the oxygen and one of the amide NH hydrogens is in a position that mimics the NH2 and N-1 of adenosine. Imidazole rotation about the glycosidic bond to the syn orientation places the amide group in a position that approximately matches the positions of the NH2 and N-3 of cytosine.
When Nucleoside 1 is substituted for any naturally occurring nucleoside, the enthalpy increases with the greatest increase for a dG substitution for the 1-C pairing (from xcex94H=74.7 (kcal/mol)/xcex94G=xe2x88x9216.5 (kcal/mol) for the G/C pairing to xcex94H=xe2x88x9245.5 (kcal/mol)/xcex94G=xe2x88x925.8 (kcal/mol)). The smallest enthalpy change occurs for a dA substitution (xcex94H=xe2x88x9272.9 (kcal/mol)/xcex94G=xe2x88x9215.4 (kcal/mol) for A/T pairing to xcex94H=xe2x88x9266.7 (kcal/mol)/xcex94G=xe2x88x9211.7 (kcal/mol) for the 1-T pairing). Correspondingly, Tm significantly decreases from 65.7xc2x0 C. and 70.5xc2x0 C. for the A-T and C-G couples, respectively, to 46.6xc2x0 C. for the 1-T pairing, 43.4xc2x0 C. for 1-G, 27.6xc2x0 C. for 1-A, and 14.6xc2x0 for 1-C.
When used in a PCR reaction, Nucleoside 1 and its N-propyl derivative are preferentially incorporated as dATP analogues (see Sala et al., Nucleic Acids Res. 24:3302, 1996). However, once incorporated into a DNA template, their ambiguous hydrogen bonding potential gave rise to misincorporation of any of the naturally occurring bases at frequencies of 3xc3x9710xe2x88x922 per base per amplification. Most of the substitutions (primarily consisting of G) were a result of rotation about the carboxamide bond when part of the template. Between 11-15% of the substitutions were due to rotation of the imidazole moiety about the glycosidic bond. As part of a DNA template, the N-propyl derivative behaved in the same way as Nucleoside 1 despite its propyl moiety. This study indicates that while Nucleoside 1 preferentially behaves as dATP, it has the ability in a PCR type environment to behave as all four naturally occurring nucleotides as well. From this and the above studies, it is evident that a wide range of duplex stability can be obtained through variations in base mimics and their placement within an oligonucleotide.
Petrruska et al., Proc. Natl. Acad. Sci. USA 85:6252-6256, 1988, have reported on the correlation between the thermodynamic stability of mismatched primers and DNA polymerase fidelity. By analyzing the melting profiles of a perfectly based paired primer with a A/T correct match at the 3xe2x80x2-end compared to primers that had either the incorrect base pair G/T, C/T, or T/T it was noted that there was a shift in free energy changes upon dissociation (xcex94xcex94G0) of 0.2, 0.3 and 0.4 kcal/mole for the terminal A/T compared to the G/T, C/T, or T/T mismatches. Interestingly, the A/T mismatch was extended (Drosphilia DNA polymerase) about 200 times faster than the G/T mismatch and about 1400 and 2500 times faster than the C/T and T/T mismatched respectively. The authors hypothesized that the binding cleft of the polymers excludes water and amplifies by amplifying free energy differences by increasing enthalpy differences in mismatched primers.
Many DNA hybridization-based diagnostic tests are being developed to identify persons who might be suffering from (or be predisposed to) specific genetic diseases (see for example, Norari et al., Gene 43:23-28, 1986) or to determine a genetic histocompatibility profile, which is useful for tissue matching between donor and patient (e.g., for a bone marrow transplant) (Sorg et al., Eur. J. Immunogen 19:391-401, 1992). However, significant problems are encountered when trying to develop simple and reliable hybridization methods using allele-specific oligonucleotide probes that differ in sequence at one nucleotide position. Norari et al. solved the mismatch hybridization problem by the addition of 10-times more unlabeled complementary oligonucleotide than the mismatched labeled oligonucleotide. However, this is an impractical solution when multiplex hybridization methods are being used.
Diagnostic tests that rely on the polymerase chain reaction (PCR) technique also experience problems associated with the hybridization of oligonucleotides. Rychlik (BioTechniques 18:84-90, 1995) examined the effects on PCR of varying the G+C content of primers at either the 5xe2x80x2 or 3xe2x80x2 end of a priming oligonucleotide. Using standard PCR buffers and conditions, oligonucleotides having a high G+C content at the 3xe2x80x2 end (the end used to extend DNA synthesis during PCR) results in high priming efficiency, but also promotes false priming due to greater tolerance for mismatches at the 5xe2x80x2 end. Moreover, the effects of mismatches in PCR are variable; mismatches located in the middle of a primer-template duplex do not significantly affect the efficiency of PCR amplification, while 3xe2x80x2-terminal base mismatches sometimes strongly affects PCR product yield. As a further complication, the strength of the effect that the various base pair mismatches have on PCR amplification is not the same as that observed for oligonucleotide hybrid formation and stability (Ikuta et al., Nucl. Acids. Res. 15:797-811, 1987; Jacobs et al., Nucl. Acids Res. 16:4637-4650, 1988).
The present invention provides methods and compositions for detecting base changes by improving the specificity and accuracy of oligonucleotide hybridization and PCR priming reactions, and further provides other related advantages.
This invention generally provides compositions and methods to increase the specificity of hybridization of nucleic acids and priming of nucleic acids in PCR.
In one aspect, the invention provides a composition comprising a nucleic acid and a salt, the salt comprising an anion and a cation, the anion selected from halogenated acetate, propionate and halogenated propionate, the cation selected from primary, secondary and tertiary ammonium comprising 1-36 carbon atoms, and quaternary ammonium comprising 4-48 carbon atoms.
In another aspect, the invention provides a composition which is non-flowing comprising an oligonucleotide of 6-100 nucleotides and a salt, the salt comprising an anion and a cation, the anion selected from acetate, halogenated acetate, propionate, and halogenated propionate, the cation selected from primary, secondary and tertiary ammonium comprising 1-36 carbon atoms, and quaternary ammonium comprising 4-48 carbon atoms.
In another aspect, the invention provides a composition which is free from organic solvent, comprising an oligonucleotide of 6-100 nucleotides and a salt, the salt comprising an anion and a cation, the anion selected from acetate, halogenated acetate, propionate, and halogenated propionate, the cation selected from primary, secondary and tertiary ammonium comprising 1-36 carbon atoms, and quaternary ammonium comprising 4-48 carbon atoms.
In another aspect, the invention provides a composition which includes a nucleic acid and a salt, the nucleic acid immobilized on a solid support, the salt .comprising an anion and a cation, the anion selected from acetate, halogenated acetate, propionate and halogenated propionate, the cation selected from primary, secondary and tertiary ammonium comprising 1-36 carbons, and quaternary ammonium comprising 4-48 carbons.
In another aspect, the invention provides a salt selected from the group:
(a) an acetate salt of a cation of the formula HN(CH3)2Ra wherein Ra is a C4-C7hydrocarbyl;
(b) a halogenated acetate salt of a cation of the formula HN(CH3)2Rb wherein Rb is a C7-C12hydrocarbyl;
(c) acetate and halogenated acetate salts of a cation of the formula H2N(C5-C7cycloalkyl)Rc where Rc is a C1-C12hydrocarbyl; and
(d) acetate and halogenated acetate salts of N-substituted piperdine, wherein the nitrogen of piperidine is substituted with C1-C12hydrocarbyl.
In another aspect, the invention provides an oligonucleotide in solution, where the oligonucleotide is formed from constituents including a plurality of fragments, each fragment shown schematically by structure (1) 
wherein, 
xe2x80x83represents a sequence of at least three nucleotides as found in wild-type DNA, where xe2x80x9cBxe2x80x9d represents a base independently selected at each location;
 represents a series of covalent chemical bonds termed a xe2x80x9cspecificity spacer,xe2x80x9d which separates and connects two bases B3 and B5;
the specificity spacer having steric and chemical properties such that
(a) it does not prevent hybridization between a fragment of structure (1) and an oligonucleotide fragment having a complementary base sequence, as shown schematically as structure (2) 
xe2x80x83and
(b) it cannot enter into hydrogen bonding with a base positioned opposite itself in a hybridized complementary base sequence of structure (2).
In another aspect, the invention provides an array which includes a plurality of oligonucleotides immobilized in an array format to a solid support, each oligonucleotide of the plurality formed from components which include a plurality of fragments, each fragment shown schematically by structure (1) 
wherein, 
xe2x80x83represents a sequence of at least three nucleotides as found in wild-type DNA, where xe2x80x9cBxe2x80x9d represents a base independently selected at each location;
 represents a series of covalent chemical bonds termed a xe2x80x9cspecificity spacer,xe2x80x9d which separates and connects two bases B3 and B5;
the specificity spacer having steric and chemical properties such that
(a) it does not prevent hybridization between a fragment of structure (1) and an oligonucleotide fragment having a complementary base sequence, as shown schematically as structure (2) 
xe2x80x83and
(b) it cannot enter into hydrogen bonding with a base positioned opposite itself in a hybridized complementary base sequence of structure (2).
In another aspect, the invention provides an oligonucleotide in solution, where the oligonucleotide is formed from components including a plurality of fragments, each fragment shown schematically by structure (1) 
wherein, 
xe2x80x83represents a sequence of at least three nucleotides as found in wild-type DNA, where xe2x80x9cBxe2x80x9d represents a base independently selected at each location;
 represents a series of covalent chemical bonds termed a xe2x80x9cspecificity spacer,xe2x80x9d which separates and connects two bases B3 and B5;
the specificity spacer having steric and chemical properties such that
(a) it does not prevent hybridization between a fragment of structure (1) and an oligonucleotide fragment having a complementary base sequence, as shown schematically as structure (2) 
(b) it enters into hydrogen bonding with a base positioned opposite itself in a hybridized complementary base sequence of structure (2); and
(c) it does not hydrogen-bond through any of adenine, guanine, cytosine, thymine or uracil.
In another aspect, the invention provides an array including a plurality of oligonucleotides immobilized in an array format to a solid support, each oligonucleotide of the plurality formed from components including a plurality of fragments, each fragment shown schematically by structure (1) 
wherein, 
xe2x80x83represents a sequence of at least three nucleotides as found in wild-type DNA, where xe2x80x9cBxe2x80x9d represents a base independently selected at each location;
 represents a series of covalent chemical bonds termed a xe2x80x9cspecificity spacer,xe2x80x9d which separates and connects two bases B3 and B5;
the specificity spacer having steric and chemical properties such that
(a) it does not prevent hybridization between a fragment of structure (1) and an oligonucleotide fragment having a complementary base sequence, as shown schematically as structure (2) 
(b) it enters into hydrogen bonding with a base positioned opposite itself in a hybridized complementary base sequence of structure (2); and
(c) it does not hydrogen-bond through any of adenine, guanine, cytosine, thymine or uracil.
The invention also provides a method of distinguishing between hybridization of a complementary nucleic acid target and a nucleic acid probe in which the probe and target are perfectly complementary and in which the probe and target have one or more base mismatches, comprising:
(a) mixing the nucleic acid target with the nucleic acid probe in a solution comprising a hybotrope;
(b) hybridizing at a discriminating temperature; and
(c) detecting probe hybridized to target, thereby determining whether the nucleic acid probe and target are perfectly complementary or mismatched.
In a preferred embodiment, the nucleic acid probe is labeled with a radioactive molecule, fluorescent molecule, mass-spectrometry tag or enzyme. In preferred embodiments, the nucleic acid probe and/or the target nucleic acid is from 6 to 40 bases. Preferably, the hybotrope is an ammonium salt as defined herein. Specific preferred ammonium salt hybotropes of the present invention include, without limitation, bis(2-methoxyethyl)amine acetate, 1 -ethylpiperidine acetate, 1-ethylpiperidine trichloroacetate, 1-ethylpiperidine trifluoroacetate, 1-methylimidizole acetate, 1-methylpiperidine acetate, 1-methylpiperidine trichloroacetate, 1-methylpyrrolidine acetate, 1-methylpyrrolidine trichloroacetate, 1-methylpyrrolidine trifluoroacetate, 2-methoxyethylamine acetate, N,N-dimethylcyclohexylamine acetate, N,N-dimethylcyclohexylamine trifluoroacetate, N,N-dimethylcyclohexylamine, N,N-dimethylheptylamine acetate, N,N-dimethylheptylamine acetate, N,N-dimethylhexylamine acetate, N,N-dimethylhexylamine acetate, N,N-dimethylisopropylamine acetate, N-ethylbutylamine acetate, N-ethylbutylamine trifluoroacetate, N,N-dimethylaminobutane trichloroacetate, N,N-dimethylisopropylamine trichloroacetate, triethanolamine acetate, triethylamine acetate, triethylamine trichloroacetate, tripropylamine acetate, and tetraethylammonium acetate. Other suitable hybotropes include LiTCA, RbTCA, GuSCN, NaSCN, NaClO4, KI, TMATCA TEATCA, TMATBA, TMTCA, TMTBA, TBATCA and TBATBA. Preferably, the hybotrope is present at a molarity of from about 0.005 M to about 6 M. Preferably, the probe nucleic acid is DNA or RNA, and the target nucleic acid is DNA or RNA. Preferably, the target nucleic acid is affixed to a solid substrate. Preferably, the method further comprises polymerase chain reaction.
The invention also provides a method of distinguishing between hybridization of a complementary nucleic acid target and a nucleic acid probe in which the probe and target are perfectly complementary and in which the probe and target have one or more base mismatches, comprising:
(a) mixing a nucleic acid target with a nucleic acid probe containing at least one abasic or deoxyNebularine substitution;
(b) hybridizing at a discriminating temperature; and
(c) detecting probe bound to the target,
thereby determining whether the nucleic acid probe and target are perfectly complementary or mismatched.
Preferably, the nucleic acid probe is labeled with a radioactive molecule, fluorescent molecule, mass-spectrometry tag or enzyme. In preferred embodiments, the nucleic acid probe is from 6 to 40 bases and/or the target nucleic acid is from 6 to 40 bases. Preferably, the method further comprises the use of a hybotrope, where the hybotrope may be an ammonium salt. Specific preferred ammonium salt hybotropes of the present invention include, without limitation, bis(2-methoxyethyl)amine acetate, 1-ethylpiperidine acetate, 1-ethylpiperidine trichloroacetate, 1-ethylpiperidine trifluoroacetate, 1-methylimidizole acetate, 1-methylpiperidine acetate, 1-methylpiperidine trichloroacetate, 1-methylpyrrolidine acetate, 1-methylpyrrolidine trichloroacetate, 1-methylpyrrolidine trifluoroacetate, 2-methoxyethylamine acetate, N,N-dimethylcyclohexylamine acetate, N,N-dimethylcyclohexylamine trifluoroacetate, N,N-dimethylcyclohexylamine, N,N-dimethylheptylamine acetate, N,N-dimethylheptylamine acetate, N,N-dimethylhexylamine acetate, N,N-dimethylhexylamine acetate, N,N-dimethylisopropylamine acetate, N-ethylbutylamine acetate, N-ethylbutylamine trifluoroacetate, N,N-dimethylaminobutane trichloroacetate, N,N-dimethylisopropylamine trichloroacetate, triethanolamine acetate, triethylamine acetate, triethylamine trichloroacetate, tripropylamine acetate, and tetraethylammonium acetate. Other suitable hybotropes include one or more of LiTCA, RbTCA, GuSCN, NaSCN, NaClO4, KI, TMATCA TEATCA, TMATBA, TMTCA, TMTBA, TBATCA and TBATBA. Preferably, the hybotrope is present at a molarity of from about 0.005 M to about 6 M. Preferably, the probe nucleic acid is DNA or RNA and the target nucleic acid is DNA or RNA. Preferably, the target nucleic acid is affixed to a solid substrate.
The invention also provides a method of increasing discrimination in a nucleic acid synthesis procedure, comprising:
(a) mixing a single-stranded nucleic acid target with an oligonucleotide primer in a solution comprising a hybotrope and a polymerase;
(b) annealing the primer to the target at a discriminating temperature; and
(c) synthesizing a complementary strand to the target to form a duplex.
Preferably, the nucleic acid primer is labeled with a radioactive molecule, fluorescent molecule, mass-spectrometry tag or enzyme. Preferably, the nucleic acid primer is from 6 to 40 bases. Preferably, the method includes using a hybotrope, where the hybotrope may be an ammonium salt. Specific preferred ammonium salt hybotropes of the present invention include, without limitation, bis(2-methoxyethyl)amine acetate, 1-ethylpiperidine acetate, 1-ethylpiperidine trichloroacetate, 1-ethylpiperidine trifluoroacetate, 1-methylimidizole acetate, 1-methylpiperidine acetate, 1-methylpiperidine trichloroacetate, 1-methylpyrrolidine acetate, 1-methylpyrrolidine trichloroacetate, 1-methylpyrrolidine trifluoroacetate, 2-methoxyethylamine acetate, N,N-dimethylcyclohexylamine acetate, N,N-dimethylcyclohexylamine trifluoroacetate, N,N-dimethylcyclohexylamine, N,N-dimethylheptylamine acetate, N,N-dimethylheptylamine acetate, N,N-dimethylhexylamine acetate, N,N-dimethylhexylamine acetate, N,N-dimethylisopropylamine acetate, N-ethylbutylamine acetate, N-ethylbutylamine trifluoroacetate, N,N-dimethylaminobutane trichloroacetate, N,N-dimethylisopropylamine trichloroacetate, triethanolamine acetate, triethylamine acetate, triethylamine trichloroacetate, tripropylamine acetate, and tetraethylammonium acetate. Other suitable hybotrope salts include LiTCA, RbTCA, GuSCN, NaSCN, NaClO4, KI, TMATCA TEATCA, TMATBA, TMTCA, TMTBA, TBATCA and TBATBA. Preferably, the hybotrope is present at a molarity of from about 0.005 M to about 6 M. Preferably, the steps of (a), (b), and (c) are repeated multiple times.
The invention also provides a method of distinguishing a single base change in a nucleic acid molecule from a wild-type sequence, comprising:
(a) mixing a single-stranded nucleic acid target with an oligonucleotide primer in a solution comprising an amine-based salt and a polymerase, wherein the oligonucleotide primer has a 3xe2x80x2-most base complementary to the wild-type sequence or the single base change;
(b) annealing the primer to the target at a discriminating temperature;
(c) extending the primer, wherein a complementary strand to the target is synthesized when the 3xe2x80x2-most base of the primer is complementary to the target; and
(d) detecting the extension of the primer.
Primer extension may be detected by methods well known in the art. For instance, direct detection of the duplex may be achieved visually using dyes, or a label may be incorporated into the primer or extension product. Suitable labels include radiolabels and fluorescent labels. The duplex may be denatured and the presence of extension product detected by any of the methods known in the art. For instance, the extension product may collected and run on a gel.